GENE REPLACEMENT THERAPY--HUMAN AND MOUSE MODELS

腺苷脱氨酶（ADA）缺乏症，一种罕见的常染色体隐性遗传疾病 导致一种严重的联合免疫缺陷，提供了一个最佳的 模型疾病，以开发疾病的基因治疗策略， 可以通过基因转移到造血组织中得到改善。我们最近 成功地在小鼠中获得人ADA的稳定长期表达， 逆转录病毒基因转移造血细胞，提供了鼓励， 该技术可进一步改进并应用于 遗传性人类疾病人类造血干细胞感染 尝试使用成功转导长期 在小鼠中的表达（Δ N2ADA），并使用新的载体， 构建了从人骨髓中富集原始干细胞 让我们能够检测这些细胞的感染性， 向量。为此，使用抗CD34单克隆抗体进行阳性选择， 抗体将与使用抗CD33的阴性选择组合， 抗Ia抗体以富集靶群体的原始 造血祖细胞原始干细胞的感染 在长期骨髓培养和通过移植 免疫缺陷小鼠中的人造血祖细胞。问题 关于安全问题和技术扩展到 将讨论血液学复溶所需的细胞。 鸟氨酸转氨甲酰酶（OTC）缺乏症，最常见和严重的 人尿素生成缺陷是一种很好的疾病模型， 测试基因治疗策略旨在纠正肝脏的先天性缺陷 细胞代谢我们最近确定了OTC的分子基础 在稀疏皮毛（spf）缺乏小鼠，小鼠模型的人 缺损逆转录病毒和腺相关病毒载体的构建将 并测试媒介的感染能力， 在体外和体内将OTC活性抑制到spf肝细胞中。的 在小肠中表达人OTC cDNA的转基因spf小鼠的产生 并且在生物化学和表型上对于OTC缺乏点是正确的 为这种疾病的基因治疗提供了新的靶组织。重组 编码OTC的逆转录病毒和腺相关病毒将用于 感染SPF小肠上皮细胞以确定基因转移是否 OTC在该组织中的表达是可能的，如果成功， OTC活性在该组织中的转导将纠正先天性缺陷 这些老鼠。

Adenosine deaminase (ADA) deficiency, a rare autosomal recessive condition causing a form of severe combined immunodeficiency, provides an optimal model disease to develop strategies for gene therapy of diseases which may be ameliorated by gene transfer into hematopoietic tissue. Our recent success in obtaining stable long term expression of human ADA in mice, via retroviral gene transfer in hematopoietic cells, offers encouragement that the techniques can be further improved and applied to gene therapy of inherited human diseases. Human hematopoietic stem cell infection will be attempted with the vector that was successful in transducing long term expression in mice (delta N2ADA) and with new vectors which will be constructed. Enrichment for primitive stem cells from hum bone marrow will allow us to examine the infectability of those cells with retroviral vectors. For this purpose, positive selection with anti-CD34 monoclonal antibodies will be combined with negative selection using anti-CD33 and anti-Ia antibodies to enrich the target population for primitive hematopoietic progenitor cells. Infection of primitive stem cells will be evaluated in long term bone marrow culture and through transplantation of human hematopoietic progenitors in immuno-deficient mice. Questions regarding issues of safety and expansion of the technology to the number of cells required for hematology reconstitution will be addressed. Ornithine transcarbamylase (OTC) deficiency, the most common and severe human defect in ureagenesis, is an excellent model disease to develop and test gene therapy strategies aimed at correcting inborn errors of liver cell metabolism. We have recently identified the molecular basis of OTC deficiency in the sparse fur (spf) mouse, the mouse model for the human defect. Construction of retroviral and adeno-associated viral vectors will be undertaken and the vectors tested for their ability to infect and transduce OTC activity into spf hepatocytes in vitro and in vivo. The creation of transgenic spf mice which express human OTC cDNA in small bowel and are biochemically and phenotypically correct for OTC deficiency points to a new target tissue for gene therapy of this disease. Recombinant retrovirus and adeno-associated virus which encode for OTC will be used to infect spf small intestinal epithelial cells to determine if gene transfer and expression of OTC in this tissue is possible and if successful transduction of OTC activity in this tissue will correct the inborn error of these mice.